We established an in-vitro model to study the mechanisms of bile reflux and cigarette smoking on the metabolic regulation of EAC (Project 2). The bile acid, deoxycholic acid (DCA), is a main component of bile reflux and thought to be the most carcinogenic. Cigarette smoking can be studied as cigarette smoke condensate (CSC). We developed an acute and chronic exposure model with DCA and CSC (Specific Research Aim 1). We hypothesize that acute metabolic and epigenetic changes may reveal dynamic, targetable events whereas the chronic exposure model will reveal longer term effects such as DNA mutations, mitochondrial DNA mutations, and changes in mitochondrial functions. We have shown that CSC and DCA increased the malignant phenotype of EAC cells. Altering mitochondrial respiration revealed a decrease in ATP production and mitochondrial membrane potential with an unexpected increase in ROS. The expression of several metabolic genes were evaluated which revealed that uncoupling protein 2 (UCP2) was significantly repressed by CSC and DCA (Specific Research Aim 2). UCP2 serves as a feedback mechanism to limit ROS in normal conditions; its reduction with CSC and DCA would explain an increase in ROS. These changes may be early events in apoptosis, so members of the Bcl-2 family were evaluated which revealed the upregulation of mitochondrial outer membrane proteins, Bak and Bax. MOMP occurs with the colocalization of Bak and Bax to the outer mitochondrial membrane. These proteins are thought to be terminal steps in apoptosis. However, at the doses in this model, no apoptosis or reduction in proliferation or viability occurred, therefore, CSC and DCA may induce MOMP but without apoptosis - a phenomenon called 'Minority MOMP' (Specific Research Aim 3). In minority MOMP, the oncogenetic proteins are released and cause DNA damage and mutagenesis instead of apoptosis. We hypothesize that minority MOMP links CSC and DCA to EAC development and progression. While Bak and Bax may lead to minority MOMP, they may paradoxically 'prime' the mitochondria for apoptosis when treated with mitochondrial inhibitors such as MitoVES. Therefore, induction of minority MOMP may be a link that enables MitoVES or other electron transport chain (ETC) inhibitors to harness the malignant changes induced by CSC and DCA to proceed past minority MOMP and result in overt apoptosis.